We have now developed cell free systems capable of carrying out the complete cycle of replication of PhiX174 DNA. In collaboration with Dr. S. Wickner, we have defined at least 12 different proteins involved in the synthesis of PhiX RFII from PhiX174 DNA. This reaction depends upon the E. coli proteins dna B, dna C(D), dna G, dna E (Pol III), dna Z, DNA binding protein, replication factors W, X, Y, Z, elongation factors I and III, Mg ions, ATP and the 4 dNTP. We have now developed PhiX infected extracts which are capable of RFI replication. These relative crude fractions require the E. coli proteins dna B, dna C(D), dna G and the rep gene product plus the PhiX gene A protein in addition to Mg ions, ATP and PhiX RFI. To date no other DNA (PM2, col EI, T3, etc.) replace PhiX RFI in the system. We plan to further disect this reaction which is rifampicin resistant, novobiocin and nalidixic acid sensitive and leads to semiconservative synthesis of RFI. We will define the model of replication. Studies with phage infected extracts show that PhiX RFI can be converted to PhiX ss 174 DNA progeny in a reaction dependent on the structural gene proteins coded for by PhiX DNA. We plan to finish up our experiments which define the proteins essential for the positive control of RNA polymerase-dependent synthesis of fd RFII; these proteins prevent PhiX DNA from being primed by RNA polymerase. BIBLIOGRAPHIC REFERENCES: Involvement of E. coli dnaZ Gene Produce in DNA Elongation in vitro, Sue Wickner and Jerard Hurwitz. Proc. Nat. Acad. Sci., 73, 1053 (1976). DNA synthesis in vitro dependent upon 0X174 replicative form I DNA. Chikako Sumida-Yasumoto, Arturo Yudelevich, and Jerard Hurwitz. Proc. Nat. Acad. Sci. 73, 1887 (1976).